High Cell Density Process for Constitutive Production of a Recombinant Phytase in Thermotolerant Methylotrophic Yeast Ogataea thermomethanolica Using Table Sugar as Carbon Source

Abstract

The yeast Ogataea thermomethanolica has recently emerged as a potential host for heterologous protein expression at elevated temperature. To evaluate the feasibility of O. thermomethanolica as heterologous host in large-scale fermentation, constitutive production of fungal phytase was investigated in fed-batch fermentation. The effect of different temperatures, substrate feeding strategies, and carbon sources on phytase production was investigated. It was found that O. thermomethanolica can grow in the temperature up to 40 °C and optimal at 34 °C. However, the maximum phytase production was observed at 30 °C and slightly decreased at 34 °C. The DOT stat control was the most efficient feeding strategy to obtain high cell density and avoid by-product formation. The table sugar can be used as an alternative substrate for phytase production in O. thermomethanolica. The highest phytase activity (134 U/mL) was obtained from table sugar at 34 °C which was 20-fold higher than batch culture (5.7 U/mL). At a higher cultivation temperature of 38 °C, table sugar can be used as a low-cost substrate for the production of phytase which was expressed with an acceptable yield (85 U/mL). Lastly, the results from this study reveal the industrial favorable benefits of employing O. thermomethanolica as a host for heterologous protein production.

Modulation of heterologous protein secretion in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 by CRISPR-Cas9 system

“…This yeast can utilize methanol or sucrose to drive the inducible expression of recombinant proteins [ 4 , 5 ]. In addition, it can be cultured to high-cell density on fed-batch fermentation using low-cost substrates, which is advantageous for industrial applications [ 6 , 7 ]. Like other yeasts, efficient secretion is a major bottleneck for heterologous protein production, in which heterologous proteins are often poorly secreted, leading to intracellular accumulation, which triggers endoplasmic reticulum (ER) stress [ 8 , 9 ].…”

Diversity and occurrence of methylotrophic yeasts used in genetic engineering

“…Studies of the suggested strain O. thermomethanolica BCC16875 continued during next following years. Pro moters typical for this train were studied (Harnpicharnchai et al, 2014;Promdonkoy et al, 2014), as well as methods of highdensity cultivation for expression of recombinant proteins (Charoenrat et al, 2016). Besides, work on optimization of strain's metabolism for increase of target products' output was started.…”

Evaluation of Methylotrophic Yeast Ogataea thermomethanolica TBRC 656 as a Heterologous Host for Production of an Animal Vaccine Candidate

“…Based on its indigenous methanol-inducible promoter and secretion signal, it has been developed as a non-conventional host for production of industrial enzymes [6,7]. The OT system has been tested for large-scale industrial enzyme production using highcell density fermentation [8,9]. Nevertheless, whether the OT system can be utilized for production of high-valued recombinant proteins of non-fungal origins remains to be demonstrated.…”